ABSTRACT
The change in the energy barriers for the heterogeneous reduction of pyruvate decarboxylase (PDC) relative to its coenzyme, thiamin pyrophosphate (ThPP), was determined experimentally using square wave voltammetry (SWV) to be 5.3 kcal/mol. These results are in agreement with those of reaction rate acceleration provided by thiamin-dependent decarboxylases relative to their coenzyme as determined kinetically based on the pK(a) suppression by the enzyme environment.
Subject(s)
Electrochemistry/methods , Pyruvate Decarboxylase/analysis , Pyruvate Decarboxylase/chemistry , Thiamine Pyrophosphate/analysis , Thiamine Pyrophosphate/chemistry , Catalysis , Enzyme Activation , Hydrogen-Ion Concentration , Isoenzymes/chemistry , KineticsABSTRACT
A capillary zone electrophoresis method for the separation and determination of thiamin and its phosphate esters (free thiamin, thiamin monophosphate, and thiamin pyrophosphate) was developed and optimized. The efficiency achieved with boric acid-sodium tetraborate decahydrate buffer (pH 8.24; 65-8 mM) at an applied potential of 30 kV gave the detection limit (S/N = 3) and the limit of quantitative measurement (S/N = 10) of thiamin and its phosphate esters ranging from 10(-4) to 6 x 10(-4) mM and from 6 x 10(-4) to 1.2 x 10(-3) mM, respectively. The effects of pH on separation and migration times of thiamin and its phosphate esters are described. The method was validated and applied to the quantitative determination of thiamin in commercial tablets containing both a massive and a normal dose of thiamin.